Donatella Perrone

Discrimination of different degrees of oral squamous cell carcinoma by means of Raman microspectroscopy and atomic force microscopy

Oral squamous cell carcinoma is a widespread cancer disease whose survival rate is strongly dependent on early diagnosis and on the degree of malignancy. The conventional histopathology methods, which are currently the standard ones for diagnosis, are very invasive so that they can be hardly proposed as screening methods for an early and accurate detection of disease. Raman microspectroscopy and atomic force microscopy can be potentially considered as useful tools for cancer diagnosis and detection of the malignancy degree because they provide information about the biochemical cellular content and nanomechanical properties, respectively, which would be modified by the onset and progression of pathology. The present work shows that both techniques can successfully discriminate the two cellular types of cells characterized by different degrees of oral squamous cell carcinoma. The discrimination by Raman microspectroscopy occurs according to a larger content of nucleic acids and a minor content of protein components in cells characterized by a larger degree of disease, whereas the discrimination by atomic force microscopy is achieved because of a decrease of stiffness as the degree of disease increases. Overall, both techniques could provide useful diagnostic information related to the degree of malignancy of the oral squamous cell carcinoma disease.

The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review)

Protein phosphorylation is an important cellular regulatory mechanism as many enzymes and receptors are activated/deactivated by phosphorylation and dephosphorylation events, by means of kinases and phosphatases. In particular, the protein kinases are responsible for cellular transduction signaling and their hyperactivity, malfunction or overexpression can be found in several diseases, mostly tumors. Therefore, it is evident that the use of kinase inhibitors can be valuable for the treatment of cancer. In this review, we discuss the mechanism of action of phosphorylation, with particular attention to the importance of phosphorylation under physiological and pathological conditions. We also discuss the possibility of using kinase inhibitors in the treatment of tumors.

Expression of salivary biomarkers in patients with oral mucositis: evaluation by SELDI-TOF/MS

OBJECTIVE This study aims to evaluate changes in proteomic salivary profile of patients with oral mucositis after adjuvant cancer treatments. MATERIALS AND METHODS Samples were collected from patients after adjuvant cancer therapies, and were analyzed by means of SELDI/TOF. Patients were separated in two groups: patients affected by mucositis (MUCOSITIS) and patient without mucositis (NO MUCOSITIS). All patients were divided in function of the anticancer treatment: patients who had radiotherapy (MUCOSITIS RADIO), had not radiotherapy (MUCOSITIS NO RADIO), had chemotherapy (MUCOSITIS CHEMO), and those who had not chemotherapy (MUCOSITIS NO CHEMO). Statistical evaluation PCA (Principal Component Analysis) was conducted with the software BIO-RAD Data Manager(™) (Version 3.5). RESULTS We found the increased peaks of 3443, 3487, and 4135 m/z in MUCOSITIS group, while 6237 m/z was reduced. These same peaks would the same modifications in MUCOSITIS RADIO, while in MUCOSITIS CHEMIO are increased 3443 and 6237 m/z but 3487, 4135 m/z are reduced. These data were confirmed by the PCA. CONCLUSION Anticancer therapy influenced the level expression of many salivary biomarkers in mucositis with a good significance. Therefore, 3443, 3487, 4135, and 6237 m/z are good biomarker candidates of oral mucositis.

Salivary proteomic signatures of oral squamous cell carcinoma

Delay in diagnosing oral squamous cell carcinoma (OSCC) can be still identified as a major cause of its high morbidity and mortality. To date, the early diagnosis for OSCC is mainly based on clinical oral examination and there is an urgent need for reliable markers; thus, advancements in molecular technologies has set the stage for investigating new markers, as well as new diagnostic matrices. The aim of the present study is to investigate the presence of proteomic signatures of OSCC in saliva and their use as potential biomarkers for early and non-invasive diagnosis. Saliva from 45 OSCC patients and 30 healthy controls was analysed by SELDI-TOF mass spectrometry and ProteinChip® technology. A supervised multivariate statistical analysis (Classification and Regression Tree - CART) was used to build models for discriminating between OSCC and controls, and between early (ES-OSCC) and late stage (LS-OSCC) cancers. The peptide with 8041 Da mass was 22-fold more expressed in OSCC, thus being a suitable potential biomarker. Classification and regression analysis allowed to build a model that was capable of correctly classifying all cancers and controls in an independent testing set, using the 8041 m/z peak as splitter. Eleven peaks were also differently expressed between ES-OSCCand LS-OSCC, but, basing on these differences, it was not possible to build an algorithm to predict tumour staging. These findings confirm that saliva proteome in OSCC patients is different from healthy controls and these variations might reflect different stages of disease progression and are worthy of further validation as diagnostic and prognostic biomarkers.

In vitro study on anti-cancer properties of genistein in tongue cancer

Purpose Tongue cancer is an extremely aggressive disease and is characterized by a poor prognosis. It is a complex disease to treat and current therapies have produced mediocre results with many side effects. Some facts suggest that natural essences can support traditional cancer therapy by carrying out a synergistic function with chemotherapy. Therefore, we evaluated the antitumor effects of genistein on tongue carcinoma cells. Methods Genistein 20, 50 and 100 µM were used for 24, 48 and 72 hours on 3 tongue carcinoma cell lines. xCELLigence system was used to evaluate the effects on cell adhesion, proliferation and to calculate IC50 values. Both MTT assay and Trypan blue assay were used to evaluate alterations in cell viability, scratch assay for cell migration and Western blot analysis for expression of some proteins. Results Cell adhesion was inhibited especially between 20 and 50 µM of genistein treatment. Proliferation was reduced by 50% for treatments with 20 µM at 24 hours, with 20 or 50 µM at 48 and 50 µM at 72 hours (P<0.0001). Viability tests confirmed a proportional reduction in concentration of genistein and duration of treatments. Even cell migration was reduced significantly (P<0.001). Genistein down-regulates vitronectin, OCT4 and survivin. Conclusion This in vitro study clarifies the anti-tumor effect of genistein on tongue carcinoma. In vivo studies are needed to confirm these data and develop a suitable delivery system that is capable of acting directly on tumor.

In vitro evaluation of the cytotoxic activity of three epoxy resin-based endodontic sealers

The aim of the study was to evaluate the cytotoxicity of three epoxy resin-based endodontic sealer, AH Plus, Sicura Seal and Top Seal. Direct and indirect cytotoxicity were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and LIVE/DEAD® Viability/Cytotoxicity Assay on MG63 osteoblasts-like cells. Data were statistically analyzed by analysis of variance and Tukey test, setting a significance level of 5%. Both results related to direct and indirect cell viability tests showed that all groups were significantly more cytotoxic than the negative control group. The cytotoxicity activity after one week of culture showed the absence of direct cytotoxicity, while a medium rate of indirect cytotoxicity. All the three epoxy resin-based sealers (AH Plus, Top Seal and Sicura Seal) showed a medium rate of cytotoxicity on osteoblasts-like cells in vitro. No significant difference was found among the sealers analyzed.

Effects of Curcumin on Squamous Cell Carcinoma of Tongue: An In Vitro Study

BACKGROUND The Squamous Cell Carcinoma of the Tongue (TSCC) is the most frequent cancer of oral cavity often characterized by poor prognosis. Conventional therapies are not very efficient and often may cause serious side effects. In this context, introduction of natural substances as possible adjuvant in the treatment and prevention of cancer is becoming a relevant topic. In fact, curcumin has been used for decades in Chinese traditional medicine for its beneficial effects. Curcumin has anticancer properties in many tumors however, its action on the tongue carcinoma is not entirely clear and many other investigations are necessary. OBJECTIVE Curcumin seems to be a good adjuvant in the treatment of head and neck tumors. However, these studies are generic and there are not many specific studies on TSCC, the most frequent and most aggressive cancer of the head-neck region. Our goal is to demonstrate its effectiveness also for TSCC. METHODS In this study, we evaluated the effects of curcumin on TSCC cells using different concentrations (1, 5, 10, 20 and 50 µM) and 3 different treatment times (24, 48 and 72 hours). The inhibition of adhesion, proliferation, viability, migration and apoptosis was studied. RESULTS IC50 value of curcumin is about 10 µM and there have been inhibitory effects even for treatments at low concentrations. Curcumin reduces migration and progression of TSCC cells and it promotes apoptosis and inhibits tumorigenesis. CONCLUSIONS These results suggest the possible use of curcumin as an anti-cancer agent in TSCC. However, in vivo studies are needed to confirm these effects and overcome its low bioavailability.

RECIPROCAL ALTERED EXPRESSION OF E-CADHERIN AND P-CADHERIN IN MUCOUS MEMBRANE PEMPHIGOID

E- and P- cadherins are involved in the selective adhesion of epidermal cells. To gain insight into the role of cadherins on the acantholysis of keratinocytes and further investigate the pathogenesis of Mucous Membrane Pemphigoid, we examined the expression of P-cadherin and E-cadherin, in normal human oral mucosa, lesional and peri-lesional mucosa in MMP. Twenty-nine samples from paraffin-embedded specimens of MMP were used for the study. Five specimens of healthy oral mucosa were evaluated as control group. To evaluate the E- and P-Cadherin expression, a mean percentage of positive cells was determined from the percentage of positive cells derived from the analysis of 100 cells in ten random areas at x400 magnification. It was observed that E-cadherin was weakly and discontinuously expressed on the epithelial layers of pemphigoid mucosa, while it was intensively expressed on all keratinocytes in normal human skin. In contrast, P-cadherin was strongly expressed throughout the entire epidermal layer in MMP samples, although its expression is restricted to the basal cell layer in normal human skin. Statistical analyses showed that the percentage of E-cadherin positive cells in the epithelium of pemphigoid cases was significantly decreased compared with that in normal human mucosa. There was a significant increase in the percentage of P-cadherin positive cells in the epithelial layers of MMP compared with normal human mucosa. The present study showed that there is downregulation of E-cadherin expression and upregulation of P-cadherin expression in MMP mucosa, which may be involved in the pathogenesis of MMP.